本文選題：SND1蛋白 + 生物信息學(xué)��； 參考：《天津醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究目的：人SND1蛋白被發(fā)現(xiàn)是一種潛在的多功能蛋白,目前已證實(shí)其在基因轉(zhuǎn)錄、RNA干擾、細(xì)胞應(yīng)激、脂肪代謝等多個(gè)生物活動(dòng)中發(fā)揮重要作用。因此,SND1蛋白表達(dá)異常勢(shì)必對(duì)其生物功能的發(fā)揮產(chǎn)生不同的影響,而目前人們對(duì)調(diào)控SND1蛋白表達(dá)的機(jī)制卻知之甚少。任何一種蛋白質(zhì)的表達(dá)都需要經(jīng)過轉(zhuǎn)錄起始、pre-mRNA的轉(zhuǎn)錄、轉(zhuǎn)錄后修飾、蛋白翻譯、翻譯后修飾等眾多精細(xì)復(fù)雜而協(xié)調(diào)有序的調(diào)控過程。本文旨在從基因轉(zhuǎn)錄起始水平探討調(diào)控人SND1蛋白表達(dá)的分子機(jī)制,并從mRNA和蛋白水平初步探討調(diào)控其表達(dá)的分子機(jī)制,進(jìn)而深入了解其表達(dá)如何影響生理或病理過程的分子機(jī)制。 方法：本課題分三部分進(jìn)行,第一部分：利用生物信息學(xué)數(shù)據(jù)庫(kù)和軟件對(duì)SND1基因特點(diǎn)進(jìn)行預(yù)測(cè)與分析,推測(cè)SND1基因啟動(dòng)子的核心區(qū)域及參與調(diào)控SND1基因轉(zhuǎn)錄起始的關(guān)鍵轉(zhuǎn)錄因子。第二部分：結(jié)合生物信息學(xué)分析結(jié)果,構(gòu)建包含SND1基因啟動(dòng)子的蟲熒光素酶質(zhì)粒,通過蟲熒光素酶活性檢測(cè)法和染色質(zhì)免疫沉淀技術(shù)對(duì)其啟動(dòng)子活性及轉(zhuǎn)錄因子的作用進(jìn)行檢測(cè)和驗(yàn)證。第三部分：通過RNA核酸印跡實(shí)驗(yàn)明確SND1的mRNA水平上是否存在多種轉(zhuǎn)錄本,其可能翻譯后形成SND1蛋白的亞型。通過GST融合蛋白釣取法檢測(cè)SND1蛋白在體外能否與自身結(jié)合形成二聚體或者多聚體,該結(jié)構(gòu)可能參與蛋白本身轉(zhuǎn)運(yùn)、儲(chǔ)存、降解等生物過程。 結(jié)果：①分析出人SND1基因的啟動(dòng)子特點(diǎn)：其啟動(dòng)子序列中缺乏典型的TATA盒結(jié)構(gòu),含有3個(gè)CAAT序列和多個(gè)GC盒。CpG島位于-650～-+295區(qū)域內(nèi)(以轉(zhuǎn)錄起始位點(diǎn)為+1,上游為正,下游為負(fù))。②成功構(gòu)建人SND1核心啟動(dòng)子及其截短片段的蟲熒光素酶質(zhì)粒,即,SND1(-493/+14)-luc和SND1(-863/-493)-luc.③轉(zhuǎn)錄因子Sp1和stat6都能增加SND1(-863/+14)-luc質(zhì)粒的蟲熒光素酶活性,其中Sp1對(duì)其表達(dá)的調(diào)控作用很強(qiáng),而stat6對(duì)其表達(dá)的調(diào)控作用較弱,都存在劑量依賴性。④轉(zhuǎn)錄因子Spl.stat6和NF-Y在體內(nèi)均能與人SND1啟動(dòng)子結(jié)合。⑤TGF-β信號(hào)傳導(dǎo)通路可能通過Spl參與調(diào)控人SND1的表達(dá),IL-4/stat6信號(hào)傳導(dǎo)通路不參與調(diào)控人SND1的表達(dá)。⑥正常和應(yīng)激狀態(tài)下,HeLa細(xì)胞和MD-MB-231細(xì)胞中人SND1的mRNA都只檢測(cè)到一個(gè)轉(zhuǎn)錄本。⑦人SND1蛋白通過SN結(jié)構(gòu)域在體外與其自身蛋白結(jié)合,而TD和TSN結(jié)構(gòu)域在體外不能與其自身蛋白結(jié)合。⑧人SND1蛋白的SNl,SN2,SN3和SN4各個(gè)單獨(dú)的結(jié)構(gòu)域均能在體外與其自身蛋白結(jié)合,SN3的結(jié)合能力最強(qiáng),SN1的結(jié)合能力最弱。 結(jié)論：①人SND1基因的啟動(dòng)子序列中缺乏典型的TATA盒結(jié)構(gòu),含有3個(gè)CAAT序列和多個(gè)GC盒。CpG島位于-650～+295區(qū)域內(nèi)(以轉(zhuǎn)錄起始位點(diǎn)為+1,上游為正,下游為負(fù))。②轉(zhuǎn)錄因子Sp1、stat6和NF-Y參與調(diào)控人SND1基因的轉(zhuǎn)錄,Sp1可能通過TGF-β信號(hào)傳導(dǎo)通路在調(diào)控過程中發(fā)揮重要作用。③正常和應(yīng)激狀態(tài)下細(xì)胞SND1的mRNA僅有一個(gè)轉(zhuǎn)錄本,不存在多個(gè)剪切狀態(tài)。④人SND1蛋白在體外通過SN結(jié)構(gòu)域與自身蛋白結(jié)合形成二聚體或者多聚體。
[Abstract]:Objective: human SND1 protein has been found to be a potential multifunctional protein. It has been proved to play an important role in many biological activities such as gene transcription, RNA interference, cell stress, fat metabolism and so on. Therefore, the abnormal expression of SND1 protein is bound to have different effects on its biological function, and people are currently regulating the SND1 eggs. The mechanism of white expression is little known. Any protein expression needs to be transcriptional starting, pre-mRNA transcription, posttranscriptional modification, protein translation, post-translational modification and so on and many sophisticated and orderly regulatory processes. This paper aims to explore the molecular mechanism of regulating the expression of SND1 protein from the gene transcriptional starting level and from M RNA and protein levels initially explore the molecular mechanisms regulating its expression, and further understand how its expression affects the molecular mechanism of physiological or pathological processes.
Methods: the subject is divided into three parts. The first part: using the bioinformatics database and software to predict and analyze the characteristics of the SND1 gene, the core region of the SND1 gene promoter and the key transcription factors involved in the regulation of the transcription initiation of the SND1 gene. The second part: the results of bioinformatics analysis and the construction of the SND1 base The promoter activity and transcription factors were detected and verified by the insect luciferase activity assay and chromatin immunoprecipitation technique. The third part: the existence of multiple transcripts on the mRNA level of SND1 by RNA nucleic acid blotting, and it may be translated into SND1 eggs after the translation. White subtype. By GST fusion protein fishing, SND1 protein can be combined with itself to form two polymer or polymer in vitro. This structure may be involved in the biological processes such as protein itself transport, storage, degradation and other biological processes.
Results: (1) the promoter characteristic of human SND1 gene was analyzed: the promoter sequence lacks typical TATA box structure, contains 3 CAAT sequences and multiple GC box.CpG islands are located in -650 ~ -+295 region (the transcription initiation site is +1, upstream is positive, and the downstream is negative). Enzyme plasmids, that is, both SND1 (-493/+14) -luc and SND1 (-863/-493) -luc. (-863/-493) -luc. 3 transcription factors Sp1 and STAT6 can increase the activity of the luciferase activity of the SND1 (-863/+14) -luc plasmid, in which the regulation of Sp1 on its expression is strong, and the regulation of the expression is weak, all stored in a dose dependent manner. Combined with human SND1 promoter. 5 TGF- beta signaling pathway may participate in the regulation of human SND1 expression through Spl, and IL-4/stat6 signaling pathway does not participate in the regulation of human SND1 expression. 6. In normal and stressful States, only one transcript is detected in HeLa cells and MD-MB-231 cells in SND1 mRNA. The TD and TSN domains can not be combined with their own proteins in vitro. The individual domains of SNl, SN2, SN3 and SN4 of SND1 protein can be combined with their own proteins in vitro. The binding ability of SN3 is the strongest, and the ability of SN1 to bind is the weakest.
Conclusion: (1) there is a lack of typical TATA box structure in the promoter sequence of human SND1 gene, which contains 3 CAAT sequences and multiple GC boxes in the region of -650 to +295 (the transcriptional starting site is +1, the upstream is positive, and the downstream is negative). The pathway plays an important role in the process of regulation. (3) there is only one transcript in the mRNA of cell SND1 in normal and stressful States, and there are no multiple shear states. (4) human SND1 protein forms a two polymer or polymer through the combination of the SN domain with its own protein in vitro.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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本文編號(hào)：2036562